A major focus of my laboratory is the structure-function correlation of chemokine receptors and mechanisms of G-protein coupled receptor (GPCR) signaling. Chemokine receptors are members of the superfamily of GPCRs and share a common three-dimensional structure composed of hepta-helical trans-membrane (TM) domains, which define multiple extra-cellular (ECL) and intra-cellular (ICL) loops. They possess extra-cellular N-termini of variable length and a C-terminal cytoplasmic domain containing unique motifs critical for ligand dependent receptor signaling, internalization and desensitization. Expression of chemokine receptors is regulated at the transcriptional level in many cell types. The architectural requirements and the need to interact precisely with cellular components pertinent to receptor function may subject them to post-translational regulatory mechanisms. Specifically, intra-cellular trafficking of some GPCRs may reflect the need for specific cellular helper proteins to facilitate maturation and transport that may underlie their tissue-specific expression. We have examined the structural elements in the carboxyl-terminal domain of CCR5 required for optimal surface expression. In the work published in 2001, we identified a membrane proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 that constituted a bi-partite motif critical for cell surface expression. Some members of the human chemokine receptor family serve as co-receptors for HIV entry besides their essential roles in regulating leukocyte chemotaxis in inflammation. M-tropic and T-tropic viruses preferentially use CCR5 (R5 strain) and CXCR4 (X4 strain) respectively. Naturally occurring mutations in the co-receptors and their cognate ligands influence HIV transmission and AIDS progression. Among CCR5 mutants, a naturally occurring 32 bp deletion, commonly observed in Caucasoid subjects and referred to as ccr5delta32 affords complete immunity in the homozygous state to M-tropic HIV infection of PBMCs. The mutant proteins are retained in the ER and not expressed at the cell surface. Epidemiological studies have linked the CCR5wt/ccr5delta32 heterozygotic state to a delay in the onset of AIDS. Two reports that addressed mechanisms underlying CCR5 deficiency in the above heterozygote concluded that the mutant proteins dominantly interfered with the functional expression of wt CCR5 by hetero-dimerization with and cytoplasmic sequestration of wt protein. We found that over-expression of CCR5 deletion mutants did not significantly affect the surface presentation or function of wt receptor. Transient physical association of CCR5 various defective mutants did not appear to be functionally relevant. We established that in the heterozygous state reduced CCR5 gene dosage rather than receptor sequestration was the major determinant of reduced CCR5 expression. Other ongoing studies address the mechanistic differences in the agonist mediated internalization and desensitization of various chemokine receptors. Agonist-driven endocytosis of chemokine receptors in primary cells and in model expression systems follows clathrin-dependent or independent itineraries. For instance, ligand mediated endocytosis of CCR5 in PBLs and monocytes was significantly slower than that of CXCR4 or CCR2B. During early times of in vitro activation of T cells, the rate of retrograde trafficking of agonist-occupied CCR5 gradually increased approaching that of CXCR4, but falling off at later times. By chemical depletion of cholesterol, by use of selective detergent extraction, and co-patching experiments, we showed significant differences in the level of raft association between CCR5 and CXCR4 or CCR2B. CCR5 signaling potential was sensitive to cholesterol depletion and paralleled the receptor sequestration sensitivity. Activation of primary cells induced fluctuations in the relative distribution of CCR5 in a temporal manner. In contrast, CXCR1, CXCR2 and CXCR4 in neutrophils were predominantly, if not exclusively embedded in non-raft domains. Using expression systems, we showed that the C-terminal domain and in particular the cysteine cluster of CCR5 was the dominant determinant of its slow endocytosis phenotype. Previously, it was shown that this domain and palmitoylation at the cysteines was required for the optimal antero-grade transport of CCR5. Palmitoylation at the cysteine clusters is crucial for optimal plasma membrane insertion. The acyl groups stabilized the raft insertion of CCR5 and coupled the agonist-occupied receptor to a caveolin dependent endocytosis. CCR5 lacking the cysteines was endocytosed via the clathrin-dependent pathway. When the C-tail of CCR5 was inserted in place of the corresponding sequence of CXCR4, the resulting chimera displayed a CCR5-like trafficking phenotype. Endocytotic itinerary of CCR5 was clathrin-independent, similar to the trafficking of raft markers CD59 and GPI linked GFP. Thus, in epithelial cells, caveolin over-expression accentuated the otherwise sluggish trafficking of raft associated CCR5. Lipid remodeling during activation that redistributes several raft-embedded receptors or activation dependent caveolin expression probably enhance CCR5 trafficking in a like manner in PBLs. Among the accessory proteins encoded by HIV-1, the 27 kDal membrane associated myristoylated Nef down-modulates the surface expression of certain cellular receptors. Our studies showed that while Nef induced modest down-modulation of cell surface density of CCR5 and CXCR4, intracellular signaling from these receptors were more severely impaired, suggesting that Nef may be subverting pathways involving secondary messengers. Nef effects on CXCR4 resulted in severe reduction in suppression of virus entry by the T-tropic HIV virus entry. In contrast Nef did not impair CCR5 usage by M-tropic HIV. This suggested that Nef might act as a HIV tropism-switching factor during natural infection. Besides its effect on cellular receptors, Nef also makes an important contribution to virulence. Nef has the potential to affect gene expression by interfering with cell-signaling pathways. Using cDNA microarray technology, we compared the patterns of cellular gene expression in the cell lines expressing various Nefs against the pattern in non-Nef expressing HeLa cells. Nef altered the expression of many cellular genes including a large subset of genes that are up regulated during T cell activation. By mimicking natural T cell activation by, Nef may optimize quiescent cells to support various stages of HIV replication.
